Mutated immunoglobulin-binding polypeptides

ABSTRACT

The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of  Staphylococcus  Protein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, wherein at least the glutamine residue at position 9 has been mutated to an amino acid other than asparagine. The invention also discloses multimers of said polypeptide, as well as separation matrices comprising the multimers or polypeptides.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of affinity chromatography,and more specifically to mutated immunoglobulin-binding domains ofProtein A, which are useful in affinity chromatography ofimmunoglobulins. The invention also relates to multimers of the mutateddomains and to separation matrices containing the mutated domains ormultimers.

BACKGROUND OF THE INVENTION

Immunoglobulins represent the most prevalent biopharmaceutical productsin either manufacture or development worldwide. The high commercialdemand for and hence value of this particular therapeutic market has ledto the emphasis being placed on pharmaceutical companies to maximize theproductivity of their respective mAb manufacturing processes whilstcontrolling the associated costs.

Affinity chromatography is used in most cases, as one of the key stepsin the purification of these immunoglobulin molecules, such asmonoclonal or polyclonal antibodies. A particularly interesting class ofaffinity reagents is proteins capable of specific binding to invariableparts of an immunoglobulin molecule, such interaction being independenton the antigen-binding specificity of the antibody. Such reagents can bewidely used for affinity chromatography recovery of immunoglobulins fromdifferent samples such as but not limited to serum or plasmapreparations or cell culture derived feed stocks. An example of such aprotein is staphylococcal protein A, containing domains capable ofbinding to the Fc and Fab portions of IgG immunoglobulins from differentspecies.

Staphylococcal protein A (SpA) based reagents have due to their highaffinity and selectivity found a widespread use in the field ofbiotechnology, e.g. in affinity chromatography for capture andpurification of antibodies as well as for detection or quantification.At present, SpA-based affinity medium probably is the most widely usedaffinity medium for isolation of monoclonal antibodies and theirfragments from different samples including industrial cell culturesupernatants. Accordingly, various matrices comprising protein A-ligandsare commercially available, for example, in the form of native protein A(e.g. Protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden) and alsocomprised of recombinant protein A (e.g. rProtein A-SEPHAROSE™, GEHealthcare). More specifically, the genetic manipulation performed inthe commercial recombinant protein A product is aimed at facilitatingthe attachment thereof to a support.

These applications, like other affinity chromatography applications,require comprehensive attention to definite removal of contaminants.Such contaminants can for example be non-eluted molecules adsorbed tothe stationary phase or matrix in a chromatographic procedure, such asnon-desired biomolecules or microorganisms, including for exampleproteins, carbohydrates, lipids, bacteria and viruses. The removal ofsuch contaminants from the matrix is usually performed after a firstelution of the desired product in order to regenerate the matrix beforesubsequent use. Such removal usually involves a procedure known ascleaning-in-place (CIP), wherein agents capable of eluting contaminantsfrom the stationary phase are used. One such class of agents often usedis alkaline solutions that are passed over said stationary phase. Atpresent the most extensively used cleaning and sanitizing agent is NaOH,and the concentration thereof can range from 0.1 up to e.g. 1 M,depending on the degree and nature of contamination. This strategy isassociated with exposing the matrix for pH-values above 13. For manyaffinity chromatography matrices containing proteinaceous affinityligands such alkaline environment is a very harsh condition andconsequently results in decreased capacities owing to instability of theligand to the high pH involved.

An extensive research has therefore been focused on the development ofengineered protein ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, Gëlich et al. (SusanneGüllich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober,Journal of Biotechnology 80 (2000), 169-178) suggested proteinengineering to improve the stability properties of a Streptococcalalbumin-binding domain (ABD) in alkaline environments. Gëlich et al.created a mutant of ABD, wherein all the four asparagine residues havebeen replaced by leucine (one residue), aspartate (two residues) andlysine (one residue). Further, Gëlich et al. report that their mutantexhibits a target protein binding behavior similar to that of the nativeprotein, and that affinity columns containing the engineered ligand showhigher binding capacities after repeated exposure to alkaline conditionsthan columns prepared using the parental non-engineered ligand. Thus, itis concluded therein that all four asparagine residues can be replacedwithout any significant effect on structure and function.

Recent work shows that changes can also be made to protein A (SpA) toeffect similar properties. US patent application publication US2005/0143566 discloses that when at least one asparagine residue ismutated to an amino acid other than glutamine or aspartic acid, themutation confers an increased chemical stability at pH-values of up toabout 13-14 compared to the parental SpA, such as the B-domain of SpA,or Protein Z, a synthetic construct derived from the B-domain of SpA(U.S. Pat. No. 5,143,844). The authors show that when these mutatedproteins are used as affinity ligands, the separation media as expectedcan better withstand cleaning procedures using alkaline agents. Furthermutations of protein A domains with the purpose of increasing the alkalistability have also been published in WO 2008/039141, JP 2006304633A, EP1992692A1, EP2202310A2, WO 2010/110288, WO 2012/086660 and WO2012/083425. However, the currently available mutations are stillsensitive to alkaline pH and the NaOH concentration during cleaning isusually limited to 0.1 M, which means that complete cleaning isdifficult to achieve. Higher NaOH concentrations, which would improvethe cleaning, lead to unacceptable capacity losses.

There is thus still a need in this field to obtain a separation matrixcontaining protein ligands having a further improved stability towardsalkaline cleaning procedures.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide a polypeptide with improvedalkaline stability. This is achieved with a polypeptide as defined inclaim 1.

One advantage is that the alkaline stability is improved over theparental polypeptides. A further advantage is a highly selective bindingtowards immunoglobulins and other Fc-containing proteins.

A second aspect of the invention is to provide a multimer with improvedalkaline stability, comprising a plurality of polypeptides. This isachieved with a multimer as defined in the claims.

A third aspect of the invention is to provide a nucleic acid or a vectorencoding a polypeptide or multimer with improved alkaline stability.This is achieved with a nucleic acid or vector as defined in the claims.

A fourth aspect of the invention is to provide an expression systemcapable of expressing a polypeptide or multimer with improved alkalinestability. This is achieved with an expression system as defined in theclaims.

A fifth aspect of the invention is to provide a separation matrixcapable of selectively binding immunoglobulins and other Fc-containingproteins and exhibiting an improved alkaline stability. This is achievedwith a separation matrix as defined in the claims.

A sixth aspect of the invention is to provide an efficient andeconomical method of isolating an immunoglobulin or other Fc-containingprotein. This is achieved with a method as defined in the claims.

Further suitable embodiments of the invention are described in thedependent claims.

Definitions

The terms “antibody” and “immunoglobulin” are used interchangeablyherein, and are understood to include also fragments of antibodies,fusion proteins comprising antibodies or antibody fragments andconjugates comprising antibodies or antibody fragments.

The terms an “Fc-binding polypeptide” and “Fc-binding protein” mean apolypeptide or protein respectively, capable of binding to thecrystallisable part (Fc) of an antibody and includes e.g. Protein A andProtein G, or any fragment or fusion protein thereof that has maintainedsaid binding property.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows results from Example 1 for the alkali stability of mutatedand non-mutated monomeric Zvar (SEQ ID NO 4) polypeptide variantscoupled to an SPR biosensor chip.

FIG. 2 shows an enlargement of FIG. 1 for the polypeptide variantshaving the highest alkali stabilities.

DETAILED DESCRIPTION OF EMBODIMENTS

In one aspect the present invention discloses a polypeptide, whichcomprises, or consists essentially of, one or more mutants of a B or Cdomain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO 1 orSEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3 or SEQ ID NO4, wherein at least the glutamine residue at position 9 has been mutatedto an amino acid other than asparagine. SEQ ID NO 4 denotes a variant ofProtein Z, here called Zvar, with the mutations N3A,N6D,N23T. Themutation of Q9 in these domains confers an improved alkali stability incomparison with the parental domain/polypeptide, without impairing theimmunoglobulin-binding properties. Hence, the polypeptide can also bedescribed as an Fc- or immunoglobulin-binding polypeptide.

SEQ ID NO 1 (SpA B domain) ADNKFNKEQQ NAFYEILHLP NLNEEQRNGF IQSLKDDPSQSANLLAEAKK LNDAQAPK SEQ ID NO 2 (SpA C domain)ADNKFNKEQQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQAPKSEQ ID NO 3 (Protein Z) VDNKFNKEQQ NAFYEILHLP NLNEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK SEQ ID NO 4 (Zvar)VDAKFDKEQQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

In some embodiments, at least the glutamine residue at position 9 of SEQID NO 1-4 has been mutated to an amino acid other than asparagine,proline or cysteine, such as to an alanine. This has the advantage thatdeamidation-sensitive asparagines are not introduced, that the chainconformation is not disturbed by introduction of prolines and no sitesfor disulfide bridges are introduced. The Q9 mutation (e.g. a Q9Amutation) may be the only mutation or the polypeptide may also comprisefurther mutations, such as in at least one of the N3, N6, Q10, E15, H18,N21, N23, N28, G/A29, D36, Q/V40, A/K42, N/E43, L/I44, E47, Q55 and P57positions. In one or more of these positions, the original amino acidresidue may e.g. be substituted with an amino acid which is notasparagine, proline or cysteine. The original amino acid residue maye.g. be substituted with an alanine, a valine, a threonine, a serine, alysine or an aspartic acid.

In certain embodiments, the amino acid residue at position 23 is athreonine or an alanine. In some embodiments, the amino acid residue atposition 3 is an alanine and/or the amino acid residue at position 6 isan aspartic acid. In certain embodiments, at least one of the amino acidresidues at position 3 and 6 is an asparagine.

In certain embodiments, the serine residue at position 33 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to a lysine. In alternative embodiments, the aminoacid residue at position 33 is a serine.

In some embodiments, the glutamine residue at position 10 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to an alanine. In alternative embodiments, the aminoacid residue at position 10 is a glutamine.

In certain embodiments, the glutamine residue at position 32 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to an alanine. In alternative embodiments, the aminoacid residue at position 32 is a glutamine.

In some embodiments, the glutamine residue at position 40 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to an alanine or valine. In alternative embodiments,the amino acid residue at position 40 is a glutamine.

In certain embodiments, the glutamine residue at position 55 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to an alanine, a serine or glutamic acid. Inalternative embodiments, the amino acid residue at position 55 is aglutamine.

In some embodiments, the amino acid residue at position 26 is aglutamine. It appears that the alkali stability of the polypeptides withQ9 mutations is better when Q26 has not been mutated to a threonine oran alanine. In alternative embodiments, the amino acid residue atposition 26 may be mutated to an amino acid other than asparagine,glutamine, proline, cysteine, threonine or alanine.

In certain embodiments, the glutamic acid residue at position 15 hasbeen mutated to an amino acid other than asparagine, glutamine, prolineor cysteine. In particular embodiments, the glutamic acid residue atposition 15 has been mutated to a lysine. This accidental mutationfurther improves the alkaline stability of the polypeptides with a Q9mutation. In alternative embodiments, the amino acid residue at position15 is a glutamic acid.

In some embodiments, the glutamic acid residue at position 47 has beenmutated to an amino acid other than asparagine, glutamine, proline orcysteine, such as to an alanine or a threonine. In alternativeembodiments, the amino acid residue at position 47 is a glutamic acid.

In some embodiments, the asparagine residue at position 21 has beenmutated to an amino acid other than glutamine, proline or cysteine, suchas to an aspartic acid. In alternative embodiments, the amino acidresidue at position 21 is an asparagine.

In certain embodiments, the aspartic acid residue at position 36 hasbeen mutated to an amino acid other than glutamine, proline or cysteine.In particular embodiments, the aspartic acid residue at position 36 hasbeen mutated to an alanine or a threonine. In alternative embodiments,the amino acid at position 36 is an aspartic acid.

In some embodiments, the mutation is selected from the group consistingof Q9A; Q9A,E15K; Q9A,E47T; Q9A,D36T; Q9A,D36A and Q9T,E47T. Thesemutations provide particularly high alkaline stabilities.

In certain embodiments, the polypeptide comprises a sequence selectedfrom the group consisting of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQID NO 9, SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22 and SEQID NO 23, and additionally SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27.It may e.g. be defined by a sequence selected from the group consistingof SEQ ID NO 6-10 and 20-23, but it may also comprise additional aminoacid residues at the C- and/or N-terminal end.

SEQ ID NO 6 Zvar(Q9A) VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK SEQ ID NO 7 Zvar(Q9A, E15K)VDAKFDKEAQ NAFYKILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 8 Zvar(Q9A, E47T) VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLATAKK LNDAQAPK SEQ ID NO 9 Zvar(Q9A, D36T)VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKTDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 10 Zvar(Q9A, D36A) VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKADPSQSANLLAEAKK LNDAQAPK SEQ ID NO 20 Zvar(Q9T, E47T)VDAKFDKETQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLATAKK LNDAQAPKSEQ ID NO 21 VDAKFDKEAQ NAFYKILHLP NLTEEQRNAF IQSLKTDPSVSKNILAAAKK LNDAQAPK SEQ ID NO 22VDNKFNKEAQ NAFYKILHLP NLTEEQRNAF IQSLKTDPSV SKNILAAAKK LNDAQAPKSEQ ID NO 23 VDNKFNKEAQ NAFYKILHLP NLTEEQRAAF IQSLKTDPSVSKNILAAAKK LNDAQAPK SEQ ID NO 24 Zvar4AQ VDAKFDKEQQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEQQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKVDAKFDKEQQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEQQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKC SEQ ID NO 25 Zvar(Q9A)4AQ VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEAQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKVDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEAQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKC SEQ ID NO 26 Zvar(Q9A, E15K)4AQ VDAKFDKEAQ NAFYKILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEAQ NAFYKILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKVDAKFDKEAQ NAFYKILHLP NLTEEQRNAF IQSLKDDPSQSANLLAEAKK LNDAQAPK VDAKFDKEAQ NAFYKILHLPNLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKC SEQ ID NO 27 Zvar(Q9A, E47T)4AQ VDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLATAKK LNDAQAPK VDAKFDKEAQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLATAKK LNDAQAPKVDAKFDKEAQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQSANLLATAKK LNDAQAPK VDAKFDKEAQ NAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SANLLATAKK LNDAQAPKC

In a second aspect the present invention discloses a multimercomprising, or consisting essentially of, a plurality of polypeptideunits as defined by any embodiment disclosed above. The multimer cane.g. be a dimer, a trimer, a tetramer, a pentamer or a hexamer. It canbe a homomultimer, where all the units in the multimer are identical orit can be a heteromultimer, where at least one unit differs from theothers. Advantageously, all the units in the multimer are alkali stable,such as by comprising the mutations disclosed above. The polypeptidescan be linked to each directly by peptide bonds between the C-terminaland N-terminal ends of the polypeptides. Alternatively, two or moreunits in the multimer can be linked by elements comprising oligomeric orpolymeric species, such as elements comprising up to 15 or 30 aminoacids, such as 1-5, 1-10 or 5-10 amino acids. The nature of such a linkshould preferably not destabilize the spatial conformation of theprotein units. This can e.g. be achieved by avoiding the presence ofproline in the links. Furthermore, said link should preferably also besufficiently stable in alkaline environments not to impair theproperties of the mutated protein units. For this purpose, it isadvantageous if the links do not contain asparagine. It can additionallybe advantageous if the links do not contain glutamine. The multimer mayfurther at the N-terminal end comprise a plurality of amino acidresidues originating from the cloning process or constituting a residuefrom a cleaved off signaling sequence. The number of additional aminoacid residues may e.g. be 15 or less, such as 10 or less or 5 or less.As a specific example, the multimer may comprise an AQ sequence at theN-terminal end.

In some embodiments, the polypeptide and/or multimer, as disclosedabove, further comprises at the C-terminal or N-terminal end one or morecoupling elements, selected from the group consisting of a cysteineresidue, a plurality of lysine residues and a plurality of histidineresidues. The coupling element may e.g. be a single cysteine at theC-terminal end. The coupling element(s) may be directly linked to the C-or N-terminal end, or it/they may be linked via a linker comprising upto 15 amino acids, such as 1-5, 1-10 or 5-10 amino acids. This stretchshould preferably also be sufficiently stable in alkaline environmentsnot to impair the properties of the mutated protein. For this purpose,it is advantageous if the stretch does not contain asparagine. It canadditionally be advantageous if the stretch does not contain glutamine.An advantage of having a C-terminal cysteine is that endpoint couplingof the protein can be achieved through reaction of the cysteine thiolwith an electrophilic group on a support. This provides excellentmobility of the coupled protein which is important for the bindingcapacity.

In a third aspect the present invention discloses a nucleic acidencoding a polypeptide or multimer according to any embodiment disclosedabove. Thus, the invention encompasses all forms of the present nucleicacid sequence such as the RNA and the DNA encoding the polypeptide ormultimer. The invention embraces a vector, such as a plasmid, which inaddition to the coding sequence comprises the required signal sequencesfor expression of the polypeptide or multimer according the invention.In one embodiment, the vector comprises nucleic acid encoding a multimeraccording to the invention, wherein the separate nucleic acids encodingeach unit may have homologous or heterologous DNA sequences.

In a fourth aspect the present invention discloses an expression system,which comprises, a nucleic acid or a vector as disclosed above. Theexpression system may e.g. be a gram-positive or gram-negativeprokaryotic host cell system, e.g. E. coli or Bacillus sp. which hasbeen modified to express the present polypeptide or multimer. In analternative embodiment, the expression system is a eukaryotic host cellsystem, such as a yeast, e.g. Pichea pastoris or Saccharomycescerevisiae.

In a fifth aspect, the present invention discloses a separation matrix,wherein a plurality of polypeptides or multimers according to anyembodiment disclosed above have been coupled to a solid support. Such amatrix is useful for separation of immunoglobulins or otherFc-containing proteins and, due to the improved alkali stability of thepolypeptides/multimers, the matrix will withstand highly alkalineconditions during cleaning, which is essential for long-term repeateduse in a bioprocess separation setting.

As the skilled person will understand, the expressed polypeptide ormultimer should be purified to an appropriate extent before beenimmobilized to a support. Such purification methods are well known inthe field, and the immobilization of protein-based ligands to supportsis easily carried out using standard methods. Suitable methods andsupports will be discussed below in more detail.

The solid support of the matrix according to the invention can be of anysuitable well-known kind. A conventional affinity separation matrix isoften of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carboxamido (—CONH₂, possibly in N-substituted forms), amino(—NH₂, possibly in substituted form), oligo- or polyethylenoxy groups ontheir external and, if present, also on internal surfaces. The solidsupport can suitably be porous. The porosity can be expressed as a Kavor Kd value (the fraction of the pore volume available to a probemolecule of a particular size) measured by inverse size exclusionchromatography, e.g. according to the methods described in GelFiltration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp6-13. By definition, both Kd and Kav values always lie within the range0-1. The Kav value can advantageously be 0.6-0.95, e.g. 0.7-0.90 or0.6-0.8, as measured with dextran of Mw 110 kDa as a probe molecule. Anadvantage of this is that the support has a large fraction of pores ableto accommodate both the polypeptides/multimers of the invention andimmunoglobulins binding to the polypeptides/multimers and to providemass transport of the immunoglobulins to and from the binding sites.

The polypeptides or multimers may be attached to the support viaconventional coupling techniques utilising e.g. thiol, amino and/orcarboxy groups present in the ligand. Bisepoxides, epichlorohydrin,CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents.Between the support and the polypeptide/multimer, a molecule known as aspacer can be introduced, which improves the availability of thepolypeptide/multimer and facilitates the chemical coupling of thepolypeptide/multimer to the support. Alternatively, thepolypeptide/multimer may be attached to the support by non-covalentbonding, such as physical adsorption or biospecific adsorption.

In some embodiments the matrix comprises 5-20, such as 5-15 mg/ml, 5-11mg/ml or 6-11 mg/ml of said polypeptide or multimer coupled to saidsupport. The amount of coupled polypeptide/multimer can be controlled bythe concentration of polypeptide/multimer used in the coupling process,by the coupling conditions used and/or by the pore structure of thesupport used. As a general rule the absolute binding capacity of thematrix increases with the amount of coupled polypeptide/multimer, atleast up to a point where the pores become significantly constricted bythe coupled polypeptide/multimer. The relative binding capacity per mgcoupled polypeptide/multimer will decrease at high coupling levels,resulting in a cost-benefit optimum within the ranges specified above.

In certain embodiments the polypeptides or multimers are coupled to thesupport via thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. Thioether bonds areflexible and stable and generally suited for use in affinitychromatography. In particular when the thioether bond is via a terminalor near-terminal cysteine residue on the polypeptide or multimer, themobility of the coupled polypeptide/multimer is enhanced which providesimproved binding capacity and binding kinetics. In some embodiments thepolypeptide/multimer is coupled via a C-terminal cysteine provided onthe protein as described above. This allows for efficient coupling ofthe cysteine thiol to electrophilic groups, e.g. epoxide groups,halohydrin groups etc. on a support, resulting in a thioether bridgecoupling.

In certain embodiments the support comprises a polyhydroxy polymer, suchas a polysaccharide. Examples of polysaccharides include e.g. dextran,starch, cellulose, pullulan, agar, agarose etc. Polysaccharides areinherently hydrophilic with low degrees of nonspecific interactions,they provide a high content of reactive (activatable) hydroxyl groupsand they are generally stable towards alkaline cleaning solutions usedin bioprocessing.

In some embodiments the support comprises agar or agarose. The supportsused in the present invention can easily be prepared according tostandard methods, such as inverse suspension gelation (S Hjertén:Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the basematrices are commercially available products, such as SEPHAROSE™ FF (GEHealthcare). In an embodiment, which is especially advantageous forlarge-scale separations, the support has been adapted to increase itsrigidity using the methods described in U.S. Pat. No. 6,602,990 or7,396,467, which are hereby incorporated by reference in their entirety,and hence renders the matrix more suitable for high flow rates.

In certain embodiments the support, such as a polysaccharide or agarosesupport, is crosslinked, such as with hydroxyalkyl ether crosslinks.Crosslinker reagents producing such crosslinks can be e.g.epihalohydrins like epichlorohydrin, diepoxides like butanedioldiglycidyl ether, allylating reagents like allyl halides or allylglycidyl ether. Crosslinking is beneficial for the rigidity of thesupport and improves the chemical stability. Hydroxyalkyl ethercrosslinks are alkali stable and do not cause significant nonspecificadsorption.

Alternatively, the solid support is based on synthetic polymers, such aspolyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L'Industria 70(9),70-75 (1988)). Alternatively, a commercially available product, such asSOURCE™ (GE Healthcare) is used. In another alternative, the solidsupport according to the invention comprises a support of inorganicnature, e.g. silica, zirconium oxide etc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter (e.g. a membrane or a depthfilter matrix).

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

In a sixth aspect, the present invention discloses a method of isolatingan immunoglobulin, wherein a separation matrix as disclosed above isused

In certain embodiments, the method comprises the steps of:

a) contacting a liquid sample comprising an immunoglobulin with aseparation matrix as disclosed above,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elutionliquid, and

d) cleaning the separation matrix with a cleaning liquid.

The method may also comprise steps of before step a) providing anaffinity separation matrix according to any of the embodiments describedabove and providing a solution comprising an immunoglobulin and at leastone other substance as a liquid sample and after step c) recovering theeluate and optionally subjecting the eluate to further separation steps,e.g. by anion or cation exchange chromatography, multimodalchromatography and/or hydrophobic interaction chromatography. Suitablecompositions of the liquid sample, the washing liquid and the elutionliquid, as well as the general conditions for performing the separationare well known in the art of affinity chromatography and in particularin the art of Protein A chromatography. The liquid sample comprising anFc-containing protein and at least one other substance may comprise hostcell proteins (HCP), such as CHO cell or E coli proteins. Contents ofCHO cell and E coli proteins can conveniently be determined byimmunoassays directed towards these proteins, e.g. the CHO HCP or E coliHCP ELISA kits from Cygnus Technologies. The host cell proteins or CHOcell/E coli proteins may be desorbed during step b).

The elution may be performed by using any suitable solution used forelution from Protein A media. This can e.g. be a solution or buffer withpH 5 or lower, such as pH 2.5-5 or 3-5. It can also in some cases be asolution or buffer with pH 11 or higher, such as pH 11-14 or pH 11-13.In some embodiments the elution buffer or the elution buffer gradientcomprises at least one mono- di- or trifunctional carboxylic acid orsalt of such a carboxylic acid. In certain embodiments the elutionbuffer or the elution buffer gradient comprises at least one anionspecies selected from the group consisting of acetate, citrate, glycine,succinate, phosphate, and formiate.

In some embodiments, the cleaning liquid is alkaline, such as with a pHof 13-14. Such solutions provide efficient cleaning of the matrix, inparticular at the upper end of the interval

In certain embodiments, the cleaning liquid comprises 0.1-2.0 M NaOH orKOH, such as 0.5-2.0 or 0.5-1.0 M NaOH or KOH.

In some embodiments, steps a)-d) are repeated at least 10 times, such asat least 50 times or 50-200 times.

EXAMPLES

Mutagenesis of Protein

Site-directed mutagenesis was performed by a two-step PCR usingoligonucleotides coding for the asparagine replacement. As template aplasmid containing a single domain of either Z or C was used. The PCRfragments were ligated into an E. coli expression vector (pGO). DNAsequencing was used to verify the correct sequence of insertedfragments. To form multimers of mutants an Acc I site located in thestarting codons (GTA GAC) of the C or Z domain was used, correspondingto amino acids VD. pGO for the monomeric domain were digested with Acc Iand CIP treated. Ace I sticky-ends primers were designed, specific foreach variant, and two overlapping PCR products were generated from eachtemplate. The PCR products were purified and the concentration wasestimated by comparing the PCR products on a 2% agarose gel. Equalamounts of the pair wise PCR products were hybridized (90° C.→25° C. in45 min) in ligation buffer. The resulting product consists approximatelyto ¼ of fragments likely to be ligated into an Acc I site (correct PCRfragments and/or the digested vector). After ligation and transformationcolonies were PCR screened to identify constructs containing the desiredmutant. Positive clones were verified by DNA sequencing.

Construct Expression and Purification

The constructs were expressed in the bacterial periplasm by fermentationof E. coli K12 in standard media. After fermentation the cells wereheat-treated to release the periplasm content into the media. Theconstructs released into the medium were recovered by microfiltrationwith a membrane having a 0.2 μm pore size.

Each construct, now in the permeate from the filtration step, waspurified by affinity. The permeate was loaded onto a chromatographymedium containing immobilized IgG. The loaded product was washed withphosphate buffered saline and eluted by lowering the pH. The elutionpool was adjusted to a neutral pH and reduced by addition ofdithiothreitol. The sample was then loaded onto an anion exchanger.After a wash step the construct was eluted in a NaCl gradient toseparate it from any contaminants. The elution pool was concentrated byultrafiltration to 40-50 mg/ml. It should be noted that the successfulaffinity purification of a construct on an immobilized IgG mediumindicates that the construct in question has a high affinity to IgG.

The purified ligands were analyzed with LC-MS to determine the purityand to ascertain that the molecular weight corresponded to the expected(based on the amino acid sequence).

Example 1

The purified monomeric ligands listed in Table 1 were immobilized onBiacore CM5 sensor chips (GE Healthcare, Sweden), using the aminecoupling kit of GE Healthcare (for carbodiimide coupling of amines onthe carboxymethyl groups on the chip) in an amount sufficient to give asignal strength of about 1000 RU in a Biacore instrument (GE Healthcare,Sweden). To follow the IgG binding capacity of the immobilized surface 1mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and thesignal strength was noted. The surface was then cleaned-in-place (CIP),i.e. flushed with 500 mM NaOH for 10 minutes at room temperature(22+/−2° C.). This was repeated for 96 cycles and the immobilized ligandalkaline stability was followed as the relative loss of IgG bindingcapacity (signal strength) after each cycle. The results are shown inFIG. 1 and Table 1 and indicate that at least the ligands Zvar(Q9A)1,Zvar(Q9A,E15K)1, Zvar(Q9A,E47T)1, Zvar(Q9A,D36T)1, Zvar(Q9A,D36A)1 andZvar(Q9T,E47T)1 have an improved alkali stability compared to theparental structure Zvar1.

TABLE 1 SEQ ID Remaining capacity Ligand NO. after 96 cycles (%) Zvar1 454 Zvar(Q9A)1 6 61 Zvar(Q9A, E15K)1 7 67 Zvar(Q9A, E47T)1 8 63 Zvar(Q9A,D36T)1 9 60 Zvar(Q9A, D36A)1 10 65 Zvar(Q9T, Q26A, E47T)1 11 5 Zvar(Q9A,Q26T, D36T, E47T)1 12 15 Zvar(Q9T, Q26T, N43T)1 13 16 Zvar(Q9T, Q26A)114 20 Zvar(Q9A, Q26T, D36T, K58R)1 15 23 Zvar(Q9T, D36A, E47T)1 16 41Zvar(Q9T, D36T)1 17 42 Zvar(Q9A, Q26A, D36A, E47A)1 18 45 Zvar(Q9A,E47T, K49E, N52S)1 19 47 Zvar(Q9T, E47T)1 20 58

Example 2

The purified tetrameric ligands of Table 2 (all with an additionalN-terminal cysteine) were immobilized on agarose beads using the methodsdescribed below and assessed for capacity and stability.

TABLE 2 Remaining Remaining Initial IgG IgG capacity IgG capacity Ligandcapacity after 100 after 100 SEQ ID content Qb10 cycles cycles LigandNO. (mg/ml) (mg/ml) (mg/ml) (%) Zvar4 24 6.4 47.6 34.1 71.7 Zvar(Q9A)425 6.3 46.3 38.8 83.9 Zvar(Q9A, 26 6.2 49.8 41.0 82.3 E15K)4 Zvar(Q9A,27 7.2 50.5 40.8 80.8 E47T)4

Activation

The base matrix used was rigid cross-linked agarose beads of 85micrometers (volume-weighted) median diameter, prepared according to themethods of U.S. Pat. No. 6,602,990 and with a pore size corresponding toan inverse gel filtration chromatography Kav value of 0.70 for dextranof Mw 110 kDa, according to the methods described in Gel FiltrationPrinciples and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.

25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 gNaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 minat 25° C. 4.0 mL of epichlorohydrin was added and the reactionprogressed for 2 hours. The activated gel was washed with 10 gelsediment volumes (GV) of water.

Coupling

To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mgNaHCO₃, 21 mg Na₂CO₃, 175 mg NaCl and 7 mg EDTA, was added. The Falcontube was placed on a roller table for 5-10 min, and then 77 mg of DTEwas added. Reduction proceeded for >45 min. The ligand solution was thendesalted on a PD10 column packed with Sephadex G-25. The ligand contentin the desalted solution was determined by measuring the 276 nm UVabsorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin U.S. Pat. No. 6,399,750. All buffers used in the experiments had beendegassed by nitrogen gas for at least 5-10 min. The ligand content ofthe gels could be controlled by varying the concentration of the ligandsolution.

After immobilization the gels were washed 3×GV with distilled water. Thegels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixedand the tubes were left in a shaking table at room temperatureovernight. The gels were then washed alternately with 3×GV {0.1 MTRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilledwater. Gel samples were sent to an external laboratory for amino acidanalysis and the ligand content (mg/ml gel) was calculated from thetotal amino acid content.

2 ml of resin was packed in TRICORN™ 5 100 columns.

Protein

Gammanorm 165 mg/ml (Octapharma), diluted to 1 mg/ml in Equilibrationbuffer.

Equilibration Buffer

APB Phosphate buffer 20 mM+0.15 M NaCl, pH 7.4 (Elsichrom AB)

Adsorption Buffer

APB Phosphate buffer 20 mM+0.15 M NaCl, pH 7.4 (Elsichrom AB).

Elution Buffers

Citrate buffer 0.1 M, pH 6.

Citrate buffer 0.1 M, pH 3.

CIP

0.5 M NaOH

The breakthrough capacity was determined with an ÄKTAExplorer 10 systemat a residence time of 2.4 minutes. Equilibration buffer was run throughthe bypass column until a stable baseline was obtained. This was doneprior to auto zeroing. Sample was applied to the column until a 100% UVsignal was obtained. Then, equilibration buffer was applied again untila stable baseline was obtained.

Sample was loaded onto the column until a UV signal of 85% of maximumabsorbance was reached. The column was then washed with equilibrationbuffer until a UV signal of 20% of maximum absorbance at flow rate 0.5ml/min. The protein was eluted with a linear gradient over 10 columnvolumes starting at pH 6.0 and ending at pH 3.0 at a flow rate of 0.5ml/min. Then the column was cleaned with 0.1M NaOH at flow rate 0.5ml/min and re-equilibrated with equilibration buffer prior to cleaningwith 20% ethanol. The last step was to check the sample concentration byloading sample through the bypass column until a 100% UV signal wasobtained.

For calculation of breakthrough capacity at 10%, the equation below wasused. That is the amount of IgG that is loaded onto the column until theconcentration of IgG in the column effluent is 10% of the IgGconcentration in the feed.

$q_{10\%} = {\frac{C_{0}}{V_{C}}\left\lbrack {V_{app} - V_{sys} - {\int_{V_{sys}}^{V_{app}}{\frac{{A(V)} - A_{sub}}{A_{100\%} - A_{sub}}*\ {dv}}}} \right\rbrack}$

A₁₀₀%=100% UV signal;

A_(sub)=absorbance contribution from non-binding IgG subclass;

A(V)=absorbance at a given applied volume;

V_(c)=column volume;

V_(app)=volume applied until 10% breakthrough;

V_(sys)=system dead volume;

C₀=feed concentration.

The dynamic binding capacity (DBC) at 10% breakthrough was calculatedand the appearance of the curve was studied. The curve was also studiedregarding binding, elution and CIP peak. The dynamic binding capacity(DBC) was calculated for 10 and 80% breakthrough.

The 10% breakthrough DBC (Qb10) was determined both before and afterrepeated exposures to alkaline cleaning solutions. Each cycle included aCIP step with 0.5 M NaOH pumped through the column at a rate of 5microliters/min, resulting in 10 min exposure time per cycle. Theexposure took place at room temperature (22+/−2° C.). Table 2 shows theremaining capacity after 100 cycles (i.e. 1000 min cumulative exposuretime to 0.5 M NaOH), both in absolute numbers and relative to theinitial capacity.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

What is claimed is:
 1. An immunoglobulin-binding polypeptide comprisinga mutant B-domain or C-domain of Staphylococcus Protein A (SpA), asspecified by SEQ ID NO:4, wherein the glutamine residue at position 9has been mutated to an alanine residue or a threonine residue, orwherein the glutamine residue at position 9 has been mutated to analanine residue or a threonine residue with further mutations in atleast one of E15 mutated to a lysine residue, D36 mutated to a threonineresidue or an alanine residue, and E47 mutated to a threonine residue;and wherein the polypeptide binds the kappa light chain of an antibodyor fragment thereof and has improved alkaline stability compared to anon-mutated B-domain or C-domain of SpA.
 2. The polypeptide of claim 1,wherein in the further mutations the original amino acid residue issubstituted at E15 with lysine.
 3. The polypeptide of claim 1, whereinin the further mutations the original amino acid residue is substitutedat D36 with threonine.
 4. The polypeptide of claim 1, wherein in thefurther mutations the original amino acid residue is substituted at D36with alanine.
 5. The polypeptide of claim 1, wherein in the furthermutations the original amino acid residue is substituted at E47 withthreonine.
 6. The polypeptide of claim 1, wherein the amino acid residueat position 9 is alanine.
 7. The polypeptide of claim 1, wherein theamino acid residue at position 9 is threonine.
 8. The polypeptide ormultimer according to claim 1, further comprising at the C-terminal orN-terminal one or more coupling elements, selected from the groupconsisting of a cysteine residue, a plurality of lysine residues and aplurality of histidine residues.
 9. A nucleic acid or a vector encodinga polypeptide or multimer according to claim
 1. 10. A separation matrix,wherein a plurality of polypeptides or multimers according to claim 1have been coupled to a solid support.
 11. A method of isolating animmunoglobulin, wherein a separation matrix according to claim 10 isused.
 12. The method of claim 11, comprising the steps of: contacting aliquid sample comprising an immunoglobulin with the separation matrix,washing said separation matrix with a washing liquid, eluting theimmunoglobulin from the separation matrix with an elution liquid, andcleaning the separation matrix with a cleaning liquid.